Project Summary: Background: Analogous to the separation of oil from water, a process called liquid-liquid phase separation (LLPS) partitions cellular contents. LLPS is dictated by intrinsic biophysical/biochemical properties of component molecules. Many phase-separated droplets form from RNA-binding proteins in complex with target RNAs. These droplets play roles in neuromuscular/neurodegenerative diseases, and cancer. In pathological contexts, the dynamics of these molecules are disrupted. It is now appreciated that RNA sequence/structure, can dictate assembly and physical state of droplets. This provides a new role for RNA sequence, which is encoding mesoscale biophysical properties of cellular bodies. There is a link between RNA sequence and droplet behaviors but the details are still a mystery. Objective/Hypothesis: To better understand how RNA structure regulates droplets, I will measure and manipulate RNA structure temporally and spatially. I will then use these measurements to gain mechanistic insights into how droplet maturation occurs and how droplets can facilitate or inhibit RNA translation. We hypothesize that sequence directed RNA structures dictates the contents, behavior, and function of condensed droplets and this in turn influences cell biology. Specific Aims: (1) To determine the role of RNA/RNA interactions in droplets. (2) To map RNA structures and RNA protein interaction changes that influence maturation of droplets. (3) To determine how RNA structures at the core and shell of droplets differ and impact functions. Study Design: I will use the model protein called Whi3 from genetically tractable fungi where RNA-structure is known to be critical to droplet formation with target RNAs. I will measure RNA/RNA interaction, RNA structure, and protein binding during the course of droplet maturation. I will then disrupt the structures and interactions identified by mutating RNA sequences, to assess the impact of these features on droplet formation, maturation, and biophysical properties (size, viscosity, diffusivity, etc.). I will next determine how the biophysical properties of the shell influence cell biological phenotypes by measuring how droplets regulate translation efficiency. Innovation: Thus far, a detailed examination of how RNA structure relates to the material and functional state of droplets has not been undertaken. The kinetics of RNA structure and protein binding are unknown, and RNA/RNA interactions in droplets have not been measured. Ultimately, this work will provide insight into the role of RNA structures in driving maturation and physical state in droplets formed from LLPS, and models developed herein will be generalizable to all RNA protein complexes. Therefore, this work contributes to a better understanding of how to manipulate RNA structures in the context of human diseases. !